The key problem in NMR spectroscopy of proteins is the resolution of individual spectral peaks and their assignments to specific nuclei in the molecule. Once this is accomplished, unique information about the structure of the protein can be derived from chemical shifts and relaxation values of the resolved peaks. One approach is to observe peaks that have chemical shifts that lie outside the normal regions of overlapping peaks such as the C2-H of histidine, certain N-H peaks, or high-field methyl peaks. We have recently resolved NMR peaks corresponding to all the histidine residues of trypsin chymotrypsin, and chymotrypsinogen. We have assigned the histidine peaks of chymotrypsin and chymotrypsinogen and the peak corresponding to the active site histidine of trypsin. Our future experiments will be designed to investigate the states of these histidine residues in various proteinases: proteinase inhibitor complexes and in chemically modified proteinases. A second approach is to use isotopic substitution to simplify NMR spectra of proteins. We have synthesized C13-labeled amino acids and are incorporating these into proteins of interest. Incorporation into the bacterial enzyme, staphylococcal nuclease, will be by biosynthesis. With the collaboration of M. Laskowski, Jr., of the Purdue Chemistry Department we have incorporated C13-labeled L-arginine into the reactive site of soybean trypsin inhibitor by an enzymatic procedure. This and other similar C13-enriched analogs of soybean trypsin inhibitor will be studied by NMR spectroscopy to determine the charged state, bonding properties, and mobility of the reactive site residue in free inhibitor and in inhibitor: proteinase complexes.